The present invention concerns isolated nucleic acid molecules encoding novel TIE ligand homologues, the TIE ligand homologue proteins encoded by such nucleic acid molecules, as well as methods and means for making and using such nucleic acid and protein molecules, and antibodies binding the disclosed TIE ligand homologues.
The abbreviations xe2x80x9cTIExe2x80x9d or xe2x80x9ctiexe2x80x9d are acronyms, which stand for xe2x80x9ctyrosine kinase containing Ig and EGF homology domainsxe2x80x9d and were coined to designate a new family of receptor tyrosine kinases which are almost exclusively expressed in vascular endothelial cells and early hemopoietic cells, and are characterized by the presence of an EGF-like domain, and extracellular folding units stabilized by intra-chain disulfide bonds, generally referred to as xe2x80x9cimmunoglobulin (IG)-likexe2x80x9d folds. A tyrosine kinase homologous cDNA fragment from human leukemia cells (tie) was described by Partanen et al., Proc. Natl. Acad. Sci. USA 87, 8913-8917 (1990). The mRNA of this human xe2x80x9ctiexe2x80x9d receptor has been detected in all human fetal and mouse embryonic tissues, and has been reported to be localized in the cardiac and vascular endothelial cells. Korhonen et al., Blood 80, 2548-2555 (1992); PCT Application Publication No. WO 93/14124 (published Jul. 22, 1993). The rat homolog of human tie, referred to as xe2x80x9ctie-1xe2x80x9d, was identified by Maisonpierre et al., Oncogene 8, 1631-1637 (1993)). Another tie receptor, designated xe2x80x9ctie-2xe2x80x9d was originally identified in rats (Dumont et al., Oncogene 8, 1293-1301 (1993)), while the human homolog of tie-2, referred to as xe2x80x9corkxe2x80x9d was descnbedin U.S. Pat. No. 5,447,860 (Ziegler). The murine homolog of tie-2 was originally termed xe2x80x9ctek.xe2x80x9d The cloning of a mouse tie-2 receptor from a brain capillary cDNA library is disclosed in PCT Application Publication No. WO 95/13387 (published May 18, 1995). The TIE receptors are believed to be actively involved in angiogenesis, and may play a role in hemopoiesis as well.
The expression cloning of human TIE-2 ligands has been described in PCT Application Publication No. WO 96/11269 (published Apr. 18, 1996) and in U.S. Pat. No. 5,521,073 (published May 28, 1996). A vector designated as xcexgt10 encoding a TIE-2 ligand named xe2x80x9chtie-2 ligand 1xe2x80x9d or xe2x80x9chTL1xe2x80x9d has been deposited under ATCC Accession No. 75928. A plasmid encoding another TIE-2 ligand designated xe2x80x9chtie-2 2xe2x80x9d or xe2x80x9chTL2xe2x80x9d is available under ATCC Accession No. 75928. This second ligand has been described as an antagonist of the TAI-2 receptor. The identification of secreted human and mouse ligands for the TIE-2 receptor has been reported by Davis et al., Cell 87, 1161-1169 (1996). The human ligand designed xe2x80x9cAngiopoietin-1xe2x80x9d, to reflect its role in angiogenesis and potential action during hemopoiesis, is the same ligand as the ligand variously designated as xe2x80x9chtie-2 1xe2x80x9d or xe2x80x9chTL-1xe2x80x9d in WO 96/11269. Angiopoietin-1 has been described to play an angiogenic role later and distinct from that of VEGF (Suri et al., Cell 87, 1171-1180 (1996)). Since TIE-2 is apparently upregulated during the pathologic angiogenesis requisite for tumor growth (Kaipainen et al., Cancer Res. 54, 6571-6577 (1994)) angiopoietin-1 has been suggested to be additionally useful for specifically targeting tumor vasculature (Davis et al., supra).
The present invention concerns novel human TIE ligand homologues with powerful effects on vasculature. The invention also provides for isolated nucleic acid molecules encoding such ligand homologues or functional derivatives thereof, and vectors containing such nucleic acid molecules. The invention further concerns host cells transformed with such nucleic acid to produce the novel TIE ligand homologues or functional derivatives thereof. The novel TIE ligand homologues may be agonists or antagonists of TIE receptors, known or hereinafter discovered. Their therapeutic or diagnostic use, including the delivery of other therapeutic or diagnostic agents to cells expressing the respective TIE receptors, is also within the scope of the present invention.
The present invention further provides for agonist or antagonist antibodies specifically binding the TIE ligand homologues herein, and the diagnostic or therapeutic use of such antibodies.
In another aspect, the invention concerns compositions comprising the novel TIE ligand homologues or antibodies specifically binding such TIE ligand homologues.
In a further aspect, the invention concerns conjugates of the novel TIE ligand homologues of the present invention with other therapeutic or cytotoxic agents, and compositions comprising such conjugates. Because the TIE-2 receptor has been reported to be upregulated during the pathologic angiogenesis that is requisite for tumor growth, the conjugates of TIE ligand homologues of the present invention to cytotoxic or other anti-tumor agents may find utility in specifically targeting tumor vasculature.
In addition, it has been found that the gene encoding at least one TIE ligand homologue herein (NL8) is amplified in certain tumor cells. Accordingly, compositions and methods for the diagnosis and treatment of tumors are also within the scope of the invention.
In yet another aspect, the invention concerns a method for identifying a cell that expresses a TIE receptor, which comprises contacting a cell with a detectably labeled TIE ligand homologue of the present invention under conditions permitting the binding of such TIE ligand homologue to the TIE receptor, and determining whether such binding has indeed occurred.
In a different aspect, the invention concerns a method for measuring the amount of a TIE ligand homologue of the present invention in a biological sample by contacting the biological sample with at least one antibody specifically binding the TIE ligand homologue, and measuring the amount of the TIE ligand homologue-antibody complex formed.
In yet another embodiment, the invention concerns a method for the inhibition of endothelial cell proliferation comprising treating endothelial cells with an effective amount of a TIE ligand homologue herein.
In a still further embodiment, the invention concerns a method for the induction of endothelial cell apoptosis comprising treating endothelial cells with an effective amount of a TIE ligand homologue herein.
In another embodiment, the invention concerns a method for determining the presence of a TIE ligand homologue by exposing a cell suspected of containing such homologue to an anti-TIE ligand homologue antibody and determining the binding of the antibody to the cell.
The invention specifically concerns a method of diagnosing tumor is a mammal, comprising detecting the level of expression of a gene encoding a TIE ligand homologue herein (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained. In a specific embodiment, the invention concerns a method of diagnosing tumor in a mammal by (a) contacting an anti-TIE ligand homologue antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-TIE ligand homologue antibody and the TIE ligand homologue in the test sample.
The invention further concerns a method for inhibiting tumor cell growth comprising exposing a cell which overexpresses an NL8 polypeptide to an effective amount of an agent inhibiting the expression and/or activity of the NL8 polypeptide.
In a further embodiment, the invention concerns an article of manufacture, comprising: (a) a container; (b) a label on the container; and (c) a composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of an NL8 polypeptide, and the active agent in the composition is an agent inhibiting the expression and/or activity of the NL8 polypeptide.
The invention further concerns a screening method for identifying polypeptide or small molecule agonists or antagonists of a TIE receptor based upon their ability to compete with a native or variant TIE ligand homologue of the present invention for binding to a corresponding TIE receptor.
The invention also concerns a method for imaging the presence of angiogenesis in wound healing, in inflammation or in tumors of human patients, which comprises administering detectably labeled TIE ligand homologues or agonist antibodies of the present invention, and detecting angiogenesis.
In another aspect, the invention concerns a method of promoting or inhibiting neovascularization in a patient by administering an effective amount of a TIE ligand homologue of the present invention in a pharmaceutically acceptable vehicle. In a preferred embodiment, the present invention concerns a method for the promotion of wound healing. In another embodiment, the invention concerns a method for promoting angiogenic processes, such as for inducing collateral vascularization in an ischemic heart or limb. In a further preferred embodiment, the invention concerns a method for inhibiting tumor growth.
In yet another aspect, the invention concerns a method of promoting bone development and/or maturation and/or growth in a patient, comprising administering to the patient an effective amount of a TIE ligand homologue of the present invention in a pharmaceutically acceptable vehicle.
In a further aspect, the invention concerns a method of promoting muscle growth and development, which comprises administering a patient in need an effective amount of a TIE ligand homologue of the present invention in a pharmaceutically acceptable vehicle.
The TIE ligand homologues of the present invention may be administered alone, or in combination with each other and/or with other therapeutic or diagnostic agents, including members of the VEGF family. Combination therapies may lead to new approaches for promoting or inhibiting neovascularization, and muscle growth and development.